STATEMENT OF INTENT Significant progress has been made in the assessment of the role of FLT3 mutations in AML. These have included analysis of FLT3-ITD expression alone, and in combination with cooperating alleles such as PML-RARa. Working with Project 1, Project 2 has been instrumental in preclinical development of FLT3 inhibitors by showing efficacy in murine models of disease, generating data from therapeutic trials in murine models of disease. In Specific Aim 1, we will explore the in vivo activity of FLT3-ITD and activation loop alleles, and try to understand the relative predilection of FLT3-ITD for myeloid lineage disease and of the FLT3 actiavtion loopalleles for lymphoid disease. We will use multiparameter flow cytometry to test the hypothesis that these alleleshave differential effect on cell fate determination at the multipotent progenitor stage (MPP or LMPP) where FLT3 is highly expressed during hematopoietic development. We will try to understand the mechanism whereby FLT3ITD, in contrast with FLT3 WT, is a potent activator of STAT5 using mutations that abrogate this activity. In Specific Aim 2, we will explore cooperating effects of these accurate genotypic models of FLT3-ITD mediated disease, working with Projects 3 and 4. These in turn will serve as useful in vivo models for testing novel combination therapies that are developed in Project 1. In Specific Aim 3, we will develop murine models of myeloproliferative disease mediated by the JAK2V617F allele, use these models to understand phenotypicpleiotropy of disease in humans, and as a platform for testing novel JAK2 inhibitors for development of clinical trials in Project 5. Overall, this is a highly interactive Project that will build on a proven track record of success and preclinical development of novel therapies for myeloid malignancies. SA 1. Generation and characterization of accurate models of leukemia mediated by mutated FLT3 using knock-in strategies. We will characterize the phenotype of each of these alleles. a. Generation and characterization of a FLT3-ITD conditional knock-in allele b. Generation and characterization of FLT3 D835Y and I836del conditional knock-in alleles c. Generation of FLT3-ITD Y589F/Y598F conditional knock-in allele that is defective in signaling to STATS SA 2. Characterize the cooperative effects of these accurate genotypic models of FLT3-ITD mediated disease with crosses to other germline alleles a. FLT3-ITD KI crossed with Cathepsin G PML-RARalpha and C/EBPalpha knock-in alleles (Interaction with Tenen Project 3) b. FLT3-ITD I836del KI crossed with MIL fusion alleles (interaction with Armstrong, Project 4) c. FLT3-ITD KI crossed with AML1-ETO conditional KI allele Use these models to test combination therapy delineated in Project 1 that could include combination signal transduction inhibitors, ATRA, HDAC inhibitors, or HSP inhibitors SA 3. Develop accurate murine models of JAK2V617F mediated MPD a. Develop and characterize a retroviral transduction model of JAK2V617F disease b. Generate and characterize JAK2V617F conditional knock-in allele c. Characterize novel potentiating mutations of JAK2V617F disease including MPL d. Characterize novel JAK2 inhibitors in murine models as developed in Project 1